scholarly journals Characterization of the Cofactors Involved in Non-Enzymatic Metmyoglobin Reduction In Vitro

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
M. Denzer ◽  
H. Comstock ◽  
C. Mowery ◽  
N. Maheswarappa ◽  
G. Mafi ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Cytochrome C ◽  
Electron Donors ◽  
Meat Color ◽  
Electron Carrier ◽  
Enzymatic Reduction ◽  
Muscle Ph ◽  
Lower Temperature ◽  
Electron Carriers

ObjectivesConsumers’ meat purchasing decisions are strongly influenced by color. Myoglobin is the primary meat pigment that contributes to meat color. Myoglobin consists of an iron-containing heme ring and amino acids in the form of globin chains. Both the state of the heme iron and the type of ligand affects meat color. The consumer-preferred bright cherry-red color oxymyoglobin is formed when the iron is in the ferrous state and oxygen bind to the heme. The oxidation of oxymyoglobin or deoxymyoglobin results in the formation of the brown color, ferric metmyoglobin. Predominant metmyoglobin accumulation negatively impacts consumer purchasing choices. Although muscle type and pre- and post-harvest factors can influence meat discoloration, meat has an inherent ability to reduce metmyoglobin through enzymatic pathways, mitochondria-mediated pathways, and non-enzymatic mechanisms. In the enzymatic pathway, an electron from NADH is transferred to metmyoglobin by an enzyme and an electron carrier; while in mitochondria-mediated pathway, an electron from the electron-transport chain is transferred via cytochromes. Previous research speculated the role of non-enzymatic pathway in meat color; however, limited studies have characterized the cofactors present in a meat system. The objectives of this study were to characterize cofactors in non-enzymatic metmyoglobin reduction and determine the effect of storage temperature and postmortem muscle pH in-vitro.Materials and MethodsPurified equine metmyoglobin was reduced in the presence of combinations of electron carriers and donors. Methylene blue and cytochrome c were evaluated as the electron carriers, and NADH and ascorbate were considered as the electron donors. The cofactors were held at 4 and 25°C to determine temperature effects on the reduction of metmyoglobin, and the same cofactor combinations were evaluated at pH of 5.2, 5.6, 6.0, and 6.4 to reflect postmortem muscle pH. Spectrophotometry was utilized to monitor the rates of metmyoglobin reduction. The experiments were replicated five times, and the data were analyzed using the Mixed Procedure of SAS.ResultsThe results indicated that methylene blue was a significantly more effective electron carrier than cytochrome c with both electron donors, ascorbate and NADH. EDTA had no impact on the non-enzymatic metmyoglobin reducing the ability of methylene blue (P = 0.91). Temperature and pH had cofactor specific effects on the non-enzymatic reduction of metmyoglobin. Lower temperature resulted in an increased non-enzymatic metmyoglobin reduction for methylene blue regardless of electron donor (ascorbate, P = 0.03, NADH, P = 0.04). As pH increased, the non-enzymatic metmyoglobin reducing activity reduced significantly in the presence of NADH and methylene blue.ConclusionIn conclusion, the characteristics of the cofactors at specific temperatures and pH impacted the non-enzymatic reduction of metmyoglobin. Further, current in vitro research indicated that non-enzymatic metmyoglobin reduction is possible at lower temperature and meat pH.

In vitro requirements for formate dehydrogenase activity from Desulfovibrio

1986 ◽  
Vol 32 (5) ◽  
pp. 425-429 ◽  
Author(s):  
Mary Ann Riederer-Henderson ◽  
Harry D. Peck Jr.
Keyword(s):  
Methylene Blue ◽  
Cytochrome C ◽  
Dehydrogenase Activity ◽  
Electron Acceptor ◽  
Formate Dehydrogenase ◽  
Direct Reduction ◽  
Protein S ◽  
Benzyl Viologen

In Desulfovibrio the protein(s) involved in formate dehydrogenase activity have not been identified or characterized. In situ assays in polyacrylamide gels demonstrated that formate dehydrogenase from either D. gigas or D. vulgaris catalyzed the direct reduction of either methylene blue or benzyl viologen in the presence of formate. Thus, the same protein was active with either electron acceptor. Although the enzyme could be stored in air without irreversible inactivation by O2, activity with either dye was stimulated by the addition of thiols to the assay mixture. In the absence of formate the thiols served as a substrate for the in situ reduction of methylene blue or benzyl viologen by the enzyme. Ammonium sulfate fractionation revealed the presence of a fraction which selectively stimulated activity with either benzyl viologen or cytochrome c3 as the electron acceptor. The stimulating fraction was nondialyzable, heat labile, and unstable upon storage. The fraction from either species could stimulate the formate dehydrogenase activity of the other species. The protein may be of physiological signficance as it increased when the cells were grown on formate, and it stimulated the formate hydrogenlyase system with cytochrome c3 as the electron carrier.

scholarly journals The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studied in vitro and with a model system of polyacrylamide films.

1982 ◽  
Vol 30 (1) ◽  
pp. 12-20 ◽  
Author(s):  
C J Van Noorden ◽  
J Tas
Keyword(s):  
Dehydrogenase Activity ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Model System ◽  
Tetrazolium Salt ◽  
Phenazine Methosulfate ◽  
Electron Carrier ◽  
Electron Carriers ◽  
Meldola Blue

The applicability of phenazine methosulfate, 1-methoxyphenazine methosulfate, menadione, and meldola blue as exogenous electron carriers for the cytochemical staining of nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent dehydrogenases has been studied quantitatively with tetranitro BT in vitro and with a model system of polyacrylamide films incorporating either purified glucose-6-phosphate dehydrogenase or intact rat liver parenchymal cells. It was found that every assay in which a tetrazolium salt is used, whether or not an electron carrier is present, has to be carried out in darkness. Menadione did not appear to be useful, because electrons were not found to be transferred directly from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) to this compound. Phenazine methosulfate at higher concentrations and meldola blue at concentrations optimal for carrying electrons to tetrazolium salts yielded a high level of "nothing dehydrogenase" activity in cell-containing films, but no inhibition of enzymatic activity was found. Factors involved in the interference of oxygen with tetrazolium salt reduction are discussed. 1-Methoxyphenazine methosulfate did not stain cellular compounds and caused only a very low nothing dehydrogenase activity. The cytochemical demonstration of dehydrogenase activity was shown to be independent on the concentration of 1-methoxyphenazine methosulfate used (50-1000 microM). It is concluded that 1-methoxyphenazine methosulfate is the exogenous electron carrier of choice.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

The warburg effect and tumour cell survival in human GBMs

2007 ◽  
Vol 30 (4) ◽  
pp. 97 ◽  
Author(s):  
A Wolf ◽  
J Mukherjee ◽  
A Guha
Keyword(s):  
Cytochrome C ◽  
Cell Survival ◽  
Expression Profile ◽  
Tumour Cell ◽  
Warburg Effect ◽  
Glycolytic Enzymes ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
The Warburg Effect

Introduction: GBMs are resistant to apoptosis induced by the hypoxic microenvironment and standard therapies including radiation and chemotherapy. We postulate that the Warburg effect, a preferential glycolytic phenotype of tumor cells even under aerobic conditions, plays a role in these aberrant pro-survival signals. In this study we quantitatively examined the expression profile of hypoxia-related glycolytic genes within pathologically- and MRI-defined “centre” and “periphery” of GBMs. We hypothesize that expression of hypoxia-induced glycolytic genes, particularly hexokinase 2 (HK2), favours cell survival and modulates resistance to tumour cell apoptosis by inhibiting the intrinsic mitochondrial apoptotic pathway. Methods: GBM patients underwent conventional T1-weighted contrast-enhanced MRI and MR spectroscopy studies on a 3.0T GE scanner, prior to stereotactic sampling (formalin and frozen) from regions which were T1-Gad enhancing (“centre”) and T2-positive, T1-Gad negative (“periphery”). Real-time qRT-PCR was performed to quantify regional gene expression of glycolytic genes including HK2. In vitro functional studies were performed in U87 and U373 GBM cell lines grown in normoxic (21% pO2) and hypoxic (< 1%pO2) conditions, transfected with HK2 siRNA followed by measurement of cell proliferation (BrdU), apoptosis (activated caspase 3/7, TUNEL, cytochrome c release) and viability (MTS assay). Results: There exists a differential expression profile of glycolytic enzymes between the hypoxic center and relatively normoxic periphery of GBMs. Under hypoxic conditions, there is increased expression of HK2 at the mitochondrial membrane in GBM cells. In vitro HK2 knockdown led to decreased cell survival and increased apoptosis via the intrinsic mitochondrial pathway, as seen by increased mitochondrial release of cytochrome-C. Conclusions: Increased expression of HK2 in the centre of GBMs promotes cell survival and confers resistance to apoptosis, as confirmed by in vitro studies. In vivo intracranial xenograft studies with injection of HK2-shRNA are currently being performed. HK2 and possibly other glycolytic enzymes may provide a target for enhanced therapeutic responsiveness thereby improving prognosis of patients with GBMs.

Mesoporous Silica Nanoparticles of Hydroxyurea: Potential

2020 ◽  
Vol 10 ◽  
Author(s):  
Kumar Nishchaya ◽  
Swatantra K.S. Kushwaha ◽  
Awani Kumar Rai
Keyword(s):  
Drug Release ◽  
Silica Nanoparticles ◽  
Mesoporous Silica Nanoparticles ◽  
Release Kinetics ◽  
Average Particle Size ◽  
Silica Nanoparticle ◽  
Average Particle ◽  
Independent Variables ◽  
Lower Temperature

Background: Present malignant cancer medicines has the advancement of magnetic nanoparticles as delivery carriers to magnetically accumulate anticancer medication in malignant growth tissue. Aim: In the present investigation, a silica nanoparticles (MSNs) stacked with hydroxyurea were combined and was optimized for dependent and independent variables. Method: In this study, microporous silica nanoparticle stacked with neoplastic medication had been prepared through emulsification followed with solvent evaporation method. Prepared MSNs were optimized for dependent and independent variables. Different formulations were prepared with varying ratio of polymer, lipid and surfactant which affects drug release and kinetics of drug release pattern. The obtained MSNs were identified by FTIR, SEM, drug entrapment, in-vitro drug release, drug release kinetics study, stability testing in order to investigate the nanoparticle characteristics. Results: The percentage drug entrapment of the drug for the formulations F1, F2, F3, was found to be 27.78%, 65.52% and 48.26%. The average particle size for F2 formulation was found to be 520 nm through SEM. The cumulative drug release for the formulations F1, F2, F3 was found to be 64.17%, 71.82% and 32.68%. The formulations were found to be stable which gives controlled drug delivery for 6 hours. Conclusion: From the stability studies data it can be culminated that formulations are most stable when stored at lower temperature or in refrigerator i.e. 5˚C ± 3˚C. It can be concluded that MSN’s loaded with hydroxyurea is a promising approach towards the management of cancer due to its sustained release and less side effects.

scholarly journals A Nitronaphthalimide Probe for Fluorescence Imaging of Hypoxia in Cancer Cells

2021 ◽  
Author(s):  
Rashmi Kumari ◽  
Vasumathy R ◽  
Dhanya Sunil ◽  
Raghumani Singh Ningthoujam ◽  
Badri Narain Pandey ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Tumor Hypoxia ◽  
Binding Pocket ◽  
Docking Studies ◽  
Single Step ◽  
Naphthalic Anhydride ◽  
Hypoxic Conditions ◽  
Fluorescence Measurements ◽  
Enzymatic Reduction

AbstractThe bioreductive enzymes typically upregulated in hypoxic tumor cells can be targeted for developing diagnostic and drug delivery applications. In this study, a new fluorescent probe 4−(6−nitro−1,3−dioxo−1H−benzo[de]isoquinolin−2(3H)−yl)benzaldehyde (NIB) based on a nitronaphthalimide skeleton that could respond to nitroreductase (NTR) overexpressed in hypoxic tumors is designed and its application in imaging tumor hypoxia is demonstrated. The docking studies revealed favourable interactions of NIB with the binding pocket of NTR-Escherichia coli. NIB, which is synthesized through a simple and single step imidation of 4−nitro−1,8−naphthalic anhydride displayed excellent reducible capacity under hypoxic conditions as evidenced from cyclic voltammetry investigations. The fluorescence measurements confirmed the formation of identical products (NIB-red) during chemical as well as NTR−aided enzymatic reduction in the presence of NADH. The potential fluorescence imaging of hypoxia based on NTR-mediated reduction of NIB is confirmed using in-vitro cell culture experiments using human breast cancer (MCF−7) cells, which displayed a significant change in the fluorescence colour and intensity at low NIB concentration within a short incubation period in hypoxic conditions. Graphical abstract

scholarly journals THE ENZYMATIC REDUCTION OF CYTOCHROME C

1940 ◽  
Vol 136 (3) ◽  
pp. 747-774 ◽  
Author(s):  
Erwin Haas ◽  
B.L. Horecker ◽  
T.R. Hogness
Keyword(s):  
Cytochrome C ◽  
Enzymatic Reduction

scholarly journals Marginal Adaptation and Micropermeability of Class II Cavities Restored with Three Different Types of Resin Composites—A Comparative Ten-Month In Vitro Study

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1660
Author(s):  
Sevda Mihailova Yantcheva
Keyword(s):  
Methylene Blue ◽  
In Vitro Study ◽  
Sem Analysis ◽  
Marginal Adaptation ◽  
Resin Composites ◽  
Polymerization Shrinkage ◽  
Gingival Margin ◽  
Different Types ◽  
Ageing Period

The development of composite materials is subject to the desire to overcome polymerization shrinkage and generated polymerization stress. An indicator characterizing the properties of restorative materials, with specific importance for preventing secondary caries, is the integrity and durability of marginal sealing. It is a reflection of the effects of polymerization shrinkage and generated stress. The present study aimed to evaluate and correlate marginal integrity and micropermeability in second-class cavities restored with three different types of composites, representing different strategies to reduce polymerization shrinkage and stress: nanocomposite, silorane, and bulk-fill composite after a ten-month ageing period. Thirty standardized class ΙΙ cavities were prepared on extracted human molars. Gingival margins were 1 mm apical to the cementoenamel junction. Cavities were randomly divided into three groups, based on the composites used: FiltekUltimate-nanocomposite; Filtek Silorane LS-silorane; SonicFill-bulk-fill composite. All specimens were subjected to thermal cycles after that, dipped in saline for 10-mounds. After ageing, samples were immersed in a 2% methylene blue. Thus prepared, they were covered directly with gold and analyzed on SEM for assessment of marginal seal. When the SEM analysis was completed, the teeth were included into epoxy blocks and cut longitudinally on three slices for each cavity. An assessment of microleakage on stereomicroscope followed. Results were statistically analyzed. For marginal seal evaluation: F.Ultimate and F.Silorane differ statistically with more excellent results than SonicFill for marginal adaptation to the gingival margin, located entirely in the dentin. For microleakage evaluation: F.Ultimate and F.Silorane differ statistically with less microleakage than SonicFill. Based on the results obtained: a strong correlation is found between excellent results for marginal adaptation to the marginal gingival ridge and micropermeability at the direction to the axial wall. We observe a more significant influence of time at the gingival margin of the cavities. There is a significant increase in the presence of marginal fissures (p = 0.001). A significant impact of time (p < 0.000) and of the material (p < 0.000) was found in the analysis of the microleakage.

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